Marek's disease (MD) is a highly contagious and oncogenic viral disease that causes immunosuppression in chickens. A molecular epidemiological investigation of Marek's disease virus (MDV) was conducted in chicken flocks in China during 2022 and 2023. Real-time PCR was used to detect MDV serotype 1 (MDV-1) in 544 clinical tissue samples. MDV-positive samples were subsequently tested for reticuloendotheliosis virus (REV), avian leukosis virus subgroups A and B (ALV-A/B), avian leukosis virus subgroup J (ALV-J), and chicken infectious anemia virus (CIAV). Full-length sequencing of the meq and pp38 genes was performed on 18 selected MDV-positive samples. The results revealed an MDV positive rate of 19.67% (107/544), with 73.83% (79/107) of these samples showing co-infection, predominantly MDV and CIAV (54.21%, 58/107). The MDV positive rates were higher in native chickens (28.43%, 29/102) and Hy-Line layers (27.62%, 29/105), whereas the co-infection rate of MDV with other avian immunosuppressive pathogens was highest in Arbor Acres broilers (91.67%, 22/24). Molecular analysis of the meq gene from circulating field MDV strains revealed typical amino acid substitutions associated with Chinese MDV isolates, including K77E, D80Y, V115A, T139A, P176R, and P217A. Notably, two additional novel substitutions, A88T and Q93R, were identified in 16 of the 18 isolates. Phylogenetic analysis indicated that the 16 isolates clustered with virulent MDV isolates from Guangdong Province (2018), whereas the remaining two grouped with virulent MDV strains circulating one decade earlier. These findings suggest that MDV infection was prevalent in chicken flocks across some regions of China during 2022 and 2023, frequently co-occurring with other avian immunosuppressive pathogens, and that virulent MDV strains with novel molecular characteristics are currently circulating in chicken populations.

Chicken infectious anemia virus (CIAV), Marek's disease virus (MDV), reticuloendotheliosis virus (REV), and avian leukosis virus (ALV) are immunosuppressive pathogens of concern for poultry. To investigate the prevalence of immunosuppressive pathogens in diseased chickens and the interactions between different viral infections, 768 tissue samples collected from diseased chickens in Jiangsu Province, China were analyzed for CIAV, MDV, REV, and ALV using polymerase chain reaction. The detection rate of these four immunosuppressive pathogens was 55.99%, and the detection rates of CIAV, MDV, REV, and ALV were 29.95, 23.05, 9.90 and 23.44%, respectively. The detection rates of coinfection, dual infection, triple infection, and quadruple infection were 23.57, 17.45, 5.47 and 0.65%, respectively. The most common dual and triple infections were CIAV + ALV (detection rate: 5.08%) and CIAV + MDV + ALV (detection rate: 2.60%). The infection of chicken flocks with immunosuppressive pathogens in Jiangsu Province showed a decreasing trend from 2016 to 2022. There was a synergistic association between CIAV and REV or CIAV and ALV (P<0.01). Therefore, infections and co-infections with immunosuppressive pathogens are prevalent in chicken flocks in Jiangsu Province, China, and CIAV plays a critical role in coinfection, providing an important guide for the future control of these diseases.

Abstract Background Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea, dehydration, vomiting, and high mortality to suckling piglets. As a strategy for antiviral therapy, artificial microRNA (amiRNA) mediated suppression of viral replication has recently become increasingly important. In this study, we evaluated the advantages of using an amiRNA vector against PEDV. Methods In this study, we evaluated the advantages of using an amiRNA vector against PEDV. We designed two single amiRNA sequences for different conserved sequences of the PEDV S and N genes, and tested their inhibitory effects on PEDV in Vero cells. Results It was obvious from the CCK-8 results that the transient transfection of amiRNA was non-toxic to the cells. In addition, our results showed that the transient expression of two amiRNAs (amiRNA-349 and amiRNA-1447) significantly reduced the expression of viral RNA and protein in the cells. The TCID50 results showed that the release of virus particles into the culture supernatant was significantly reduced, with an effect as high as 90%. To avoid virus mutation escape, the above two single amiRNA sequences were tandem in this study (amiRNA-349 + 1447), enabling a single microRNA to be expressed simultaneously. The real-time PCR and Western blot results showed that the inhibitory effect was significantly enhanced in each of the different time periods. The TCID50 results showed that the release of virus particles in the culture supernatant was significantly reduced at the different time periods. Conclusions In summary, these results suggest that an RNAi based on amiRNA targeting the conserved region of the virus is an effective method to improve PEDV nucleic acid inhibitors and provide a novel treatment strategy for PEDV infection.

为提高高校实验室的管理水平和技术能力,出具具有法律效力的检测报告,扬州大学动物疾病检测与技术服务中心于 2018 年 1 月通过江苏省CMA认证,成为江苏高校系统动物疾病检测领域首个通过CMA认证的第三方检测实验室.依据资质认定的最新要求,结合高校实验室工作的实际情况,从中心资质认定体系的建立、试运行、现场评审及正式运行四个方面,系统阐述了高校动检实验室资质认定体系建成的整个过程,对高校检测实验室的建设具有一定的意义.

为明确江苏省高邮市某鸡场商品蛋鸡感染疾病情况,对患病商品蛋鸡病料进行禽腺病毒(fowl adenovirus,FAdV)、马立克病毒(Marek's disease virus,MDV)、禽白血病病毒(avian leukosis virus,ALV)核酸检测和细菌分离鉴定.结果显示:该鸡场患病商品蛋鸡为禽腺病毒(D种血清 11 型)和沙门菌混合感染;沙门菌分离株对利福平、恩诺沙星、链霉素、庆大霉素、氨苄西林和多西环素耐药,对环丙沙星、氟苯尼考、头孢曲松、阿米卡星、四环素、磷霉素、头孢噻肟、卡那霉素、新霉素、呋喃唑酮、氯霉素和大观霉素敏感.

2019年10月21日—12月中旬,江苏某动物园小熊猫(Ailurus fulgens)出现疑似犬瘟热病毒(Canine distemper virus,CDV)感染,用犬瘟热胶体金试纸条检测,结果为弱阳性,共12只小熊猫死亡.对病死小熊猫剖检,可见明显的肺充血、出血,肝肿大,心内膜出血,胃肠黏膜出血.采集2只发病小熊猫尾静脉血送检,RT-PCR检测犬瘟热核酸为阳性,用Vero-CD150细胞对样品进行病毒分离,在Vero细胞上连续传代,通过RT-PCR检测,病毒核酸仍然为阳性,最终分离到2株犬瘟热病毒,分别命名为SZ2019CDV1H和SZ2019CDV2H.扩增H基因并测序,与GenBank已公布的基因序列进行同源性比较和遗传发生分析,SZ2019CDV1H株与SZ2019CDV2H株的基因同源性为97.2％.在遗传发生关系树上,SZ2019CDV1H株为Africa毒株,SZ2019CDV2H株为Eu-ropean wildlife毒株,分属于不同的遗传分支.

Rothia nasimurium was known previously as an opportunistic pathogen of animals. However, there are few reports regarding the pathogenicity of Rothia nasimurium. In September 2020, geese contracted a disease of unknown cause which brought economic losses to a farm in Jiangsu Province, China, prompting a series of investigations. The bacterium was isolated, cultured, and purified, and then identified using Gram staining, biochemical tests, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and 16S rRNA sequence analysis. After determining the obtained bacteria species, antibiotic susceptibility tests and animal regression experiments were carried out. A strain of bacterium was successfully isolated from the livers of the diseased geese, which was identified as a strain of the Gram-positive bacterium Rothia nasimurium according to the 16S rRNA sequencing results. By indexing references, no goose was reported to have been infected with Rothia nasimurium. The antibiotic susceptibility testing showed that only four antibiotics (amikacin, cefazolin, fosfomycin, and ampicillin/sulbactam) could effectively inhibit the growth of the Rothia nasimurium strain. The animal regression experiments showed that the novel isolated strain could infect goslings, and it also causes serious depilation of goslings. The results of the manuscript expanded the range of pathogenic microorganisms in geese, which is helpful to develop methods for avian endemic control.

[目的]查明江苏某鹅场鹅发病及死亡原因.[方法]剖检病死鹅,运用细菌分离纯化、染色观察、生化试验、基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)鉴定、16S rDNA测定方法进行病原菌分离鉴定,通过药敏试验和小鼠致病性试验探究分离菌株的特性.[结果]从患病鹅肝脏分离到1株革兰氏阴性短杆状细菌;生化试验结果显示,此菌株可发酵葡萄糖、麦芽糖、枸橼酸盐、尿素;MALDI-TOF MS鉴定结果显示,此菌株为类志贺邻单胞菌;16S rDNA序列对比分析发现,分离菌与类志贺邻单胞菌相似性最高,达98％以上;药敏试验结果显示,分离菌对左氧氟沙星、美罗培南、头孢西丁、庆大霉素等10种抗菌药敏感,对四环素、头孢吡肟、头孢他啉、头孢唑林表现为中介,对阿奇霉素、复方新诺明、氨苄西林等6种药物表现为耐药,是典型的多重耐药菌;小鼠致病性试验结果显示,分离菌对小鼠的半数致死量为5.0×106.5 CFU.[结论]本试验首次报道了从鹅体内分离到致病性类志贺邻单胞菌,通过药敏试验筛选了左氧氟沙星、美罗培南、头孢西丁等有效的临床常用抗菌药,分离株对小鼠致病性较强,提示其带来的潜在风险不可忽视.

2022年4月,江苏淮安某鸡场出现疑似病毒和细菌混合感染病例,为确定病原感染情况,笔者对送检的病死商品蛋鸡进行病理剖检、病毒核酸检测及细菌分离鉴定.结果显示:该鸡场鸡只存在禽白血病病毒和大肠杆菌的混合感染情况,且大肠杆菌分离株对临床常用药物耐药性强,仅对磷霉素、多黏菌素和大观霉素敏感.

Background: Porcine epidemic diarrhea (PED) is an acute and highly contagious infectious disease caused by the porcine epidemic diarrhea virus (PEDV) that occurs most frequently from winter to spring. It is associated with high morbidity and mortality rates, especially among piglets, and causes huge losses in the pig industry. The aim of this molecular epidemiological study was to identify the current strains of PEDV that are prevalent in Jiangsu Province, China.Methods: From winter 2020 to spring 2021, 793 small intestine tissue, fecal, and anal swab samples were collected from 72 pig farms in 11 counties in the jurisdiction of 5 regions of Jiangsu Province (Yancheng, Suqian, Changzhou, Xuzhou, and Yangzhou). A highly variable region of the S gene was amplified and sequenced, and phylogenetic analysis was conducted to compare this sequence with corresponding sequences from reference strains deposited in GenBank. Results: A total of 457 samples from 57 pig farms were positive for PEDV: this implies a positivity rate of 79% (57/72) for pig farms and a sample positivity rate of 57.6% (457/793). The positivity rates were 78% (107/137) in Yancheng, 53% (218/409) in Suqian, 48% (94/195) in Changzhou, 80% (16/20) in Xuzhou, and 88% (14/16) in Yangzhou. Seven representative samples were selected for sequencing, and phylogenetic analysis showed that the seven isolated strains exhibited 88.0%–100% nucleotide identity and 87.3%–99% amino acid identity. Additionally, our isolates exhibited 88.3%–99.7% nucleotide identity and 88%–98.5% amino acid identity with the reference PEDV strains. Phylogenetic tree analysis indicated that there were considerable difference in the sources of the variants.Conclusions: PEDV had a high infection rate among pigs and is possibly the main pathogenic agent of pig diarrhea in Jiangsu province. Importantly, vaccines must be screened for their efficacy against the newly identified variants.

Chicken infectious anemia virus (CIAV) mainly infects chickens and causes immunosuppression. In this study, a CIAV isolate, designated as Pigeon-CIAV-1906, was efficiently isolated from two sick pigeons by inoculating the samples into MSB1 cells. The genome of Pigeon-CIAV-1906 was amplified by PCR and analyzed. The genome size of Pigeon-CIAV-1906 was 2,298 bp with the highest homology (99.5%) to Jilin strain (JL14023) and the lowest homology (91.5%) to Brazil strain (KY024579), which phylogenetically clustered into Group A. Notably, several amino acids such as 139K and 394Q related with high virulence were found in the VP1 of Pigeon-CIAV-1906. The isolation of Pigeon-CIAV-1906 and its molecular characteristics provide evidence for the cross-transmission of CIAV from chicken to pigeon and give novel insights into the molecular epidemiology of CIAV.

本研究旨在制备非洲猪瘟病毒(ASFV)S273R蛋白的特异性单克隆抗体.本研究以原核表达的非洲猪瘟病毒重组S273R蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞.结果显示:基于纯化的S273R蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了 4株可稳定分泌抗ASFV S273R蛋白单克隆抗体的杂交瘤细胞株,并通过免疫印迹(Western blot)和免疫荧光试验(immunofluorescence assay,IFA)测定了其免疫特性.4株单克隆抗体亚类均为IgG1型,轻链均为κ链.本研究获得的非洲猪瘟病毒S373R蛋白单克隆抗体可为进一步研究ASFV pS273R的生物学功能及基因缺失毒株的鉴别诊断和疫苗开发提供技术储备.

A型塞内卡病毒(SVA)是一种单股正链RNA病毒,属于微RNA病毒科(Picornaviridae)塞内卡病毒属(Senecavirus).SVA作为猪水疱病的一种新病原体,在多个国家传播和发展迅速,引发猪不同程度的水疱性疾病,尤其以巴西、中国以及越南等多个国家病情较为严重,给当地养猪业造成重大的经济损失.该病原导致的临床症状与口蹄疫相似,且不同性别和年龄阶段的猪均易感,可引起感染猪的鼻吻、蹄冠部出现水疱性病变,仔猪急性死亡.因此,需要对SVA的流行和发病引起足够的重视,并通过一系列快速检测方法进行监测以防止该病毒的传播.本文对目前A型塞内卡病毒检测的技术和方法进行了综述,并对其应用情况以及各自的优、缺点进行了分析和总结,以期为A型塞内卡病毒检测方法的深入研究提供参考.

Background: Goose parvoviruses (GPVs) spread globally and cause a huge economic loss to the poultry industry. Although the attenuated GPV vaccines play a key role in preventing the disease caused by GPV, the molecular basis for the attenuation of GPV is barely known. Results: A highly attenuated GPV strain, GPV-CZM-142, was generated through blindly passaging of the highly pathogenic strain, GPV-CZM, in goose embryonic fibroblasts (GEF) for 142 generations. The GEF-adapted GPV strain's virulence was 10,000 times weaker than its wild type counterpart, GPV-CZM, based on the ELD 50 (50% Embryo Lethal Dose). By comparing with the wild type strain, genome sequencing analysis identified adapted mutations either in ITR or in NS and VP1 of GPV-CZM-142. Conclusions: The highly attenuated GPV strain, GPV-CZM-142, provides a GPV vaccine candidate, and the identified virulence-related mutations give a novel insight into the molecular determinants of GPV virulence.

An electrochemical immunosensor for the determination of porcine epidemic diarrhea virus (PEDV) is described. It was manufactured by using gold nanoparticles/molybdenum disulfide/reduced graphene oxide nanocomposites modified on the surface of a glassy carbon electrode (GCE). The independently developed monoclonal antibody of PEDV-2C11 was immobilized on the modified electrode at site of gold nanoparticles provided in the nanocomposites. The concentration of PEDV was quantified by measuring the changes in the charge transfer resistance of the electrode before and after the immunoreaction between antigen-antibody by using hexacyanoferrate(II)/(III) as the redox probe. The frequency range was 10−1 to 105 Hz at the amplitude of 10 mV and an applied potential of + 0.180 V. Based on the immunoreaction between PEDV antigen and PEDV-2C11 antibody in 0.1 M phosphate buffer containing 0.1 M KCl at 37.5 °C for 140 min, the relative change in impedance was proportional to the logarithmic value of PEDV concentrations in the range of 82.5 to 1.65 × 104 TCID50 mL−1. Good reproducibility, stability, and specificity of the proposed immunosensor were obtained. It was successfully applied to the determination of PEDV in the spiked sample.

采用沉淀法制备硫化镉纳米晶体,并与氨水混合加热制备纳米硫化镉-氨水复合物(CdS-NH3 NCs),将其修饰于玻碳电极后通过戊二醛将猪流行性腹泻病毒的抗体PEDV-2C11固定于电极表面制成电化学发光免疫传感器,实现了对猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)的定量检测.实验结果表明:当PEDV-2C11抗体与PEDV发生免疫反应时,CdS-NH3与过硫酸根之间的电化学发光反应电子转移受阻,使得电化学发光强度降低;在最佳实验条件下,发光强度变化与PEDV浓度在1.65～1.65×105 TCID50·mL-1范围内呈良好的线性关系.该方法成功应用于样品中PEDV的检测,表明该电化学发光免疫传感器对猪腹泻病的临床诊断具有重要意义.

随着社会的需求,第三方检验检测机构的数量日益增多.检测数据的准确性不仅关乎检测机构的检测水平,也直接影响行政机关的执法效力,因此检测实验室必须确保其检测数据科学公正、准确可靠.核酸检测是第三方检验检测机构中最常见的检测项目,而气溶胶污染是核酸检测中最主要最常见的污染,也是困扰绝大多数实验室的难题,必须要加以重视.本文就本中心在核酸检测防污染方面的体会进行初步总结.

Prototheca is a widespread microalgae,which belongs to Chlorococcum sp.People and animal can commomly infect prototheca.Among of them,Prototheca zopfii was related to severe mastitis of cow.Milk samples of cow having mastitis were collected from part of Jiangsu.4 P.zopfii were identified by gram stain microscopy and PCR method using sheep blood agar to cultur and isolate them.P.zopfii sourced from cowed was first discovered in Jiangsu area.

研究以10日龄死亡雏鸡体内分离的一株肺炎克雷伯菌为研究对象,纸片扩散法药敏结果显示对氨苄青霉素、先锋霉素V、氨苄西林/舒巴坦、头孢噻肟、复方新诺明、氨曲南、氯霉素、四环素、诺氟沙星等9种药物具有抗性.对其携带基因型进行PCR鉴定显示该菌株为产CTX-M-27型超广谱β-内酰胺酶(Extended-spectrum β-1actamases,ESBLs)菌株,并且携带Ⅰ型整合酶,此外还检测到了SHV-26、TEM-1型β-内酰胺酶.研究结果为防治临床动物感染产ESBLs肺炎克雷伯菌提供了参考,同时也揭示了ESBLs菌耐药遗传机制.

将从临床采集的发病鸡组织处理后经绒尿膜接种7日龄SPF鸡胚,通过PCR方法对鸡胚培养物进行鉴定,分离到3株禽腺病毒.结合免疫学实验、病理学实验以及动物回归实验对病毒进行特性研究,结果表明分离的病毒均能致鸡死亡并表现出临床症状.使用针对Ⅰ群禽腺病毒Hexon基因的特异性引物对病毒进行基因测序分析发现,分离株均为禽腺病毒基因E型血清8b型.